Antibody recognizing a small subset of human hematopoietic cells

ABSTRACT

The subject invention pertains to antibodies that have binding specificity for an antigen that is expressed on a subset of human, hematopoietic mononuclear cells, including a hematopoietic stem cell population, but is not expressed on normal, mature myeloid cells. In one embodiment, a monoclonal antibody, MG1, is provided. This antibody is useful in methods of isolating cell suspensions from human blood and marrow that can be employed in bone marrow transplantation, genetic therapy, and in treating other diseases of the hematopoietic system. Cell suspensions containing MG1 +  human hematopoietic cells are also provided, as well as therapeutic methods employing the cell suspensions. The subject invention also pertains to the novel antigen recognized by the subject antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/299,048,filed Nov. 18, 2002, now U.S. Pat. No. 6,838,282, which is a division ofU.S. application Ser. No. 09/873,835, filed Jun. 4, 2001, now U.S. Pat.No. 6,482,598, which is a division of U.S. application Ser. No.09/531,827, filed Mar. 21, 2000, now U.S. Pat. No. 6,242,579, which is adivision of U.S. application Ser. No. 08/970,032, filed Nov. 13, 1997,now U.S. Pat. No. 6,043,348, which claims the benefit of provisionalapplication U.S. Ser. No. 60/030,428, filed Nov. 13, 1996.

FIELD OF THE INVENTION

The present invention relates to a novel monoclonal antibody recognizinga small subset of human hematopoietic cells, which may include thehematopoietic stem cell population.

BACKGROUND OF THE INVENTION

Hematopoietic stem cells are defined as those cells that are capable ofboth self-renewal and differentiation into the two principle precursorcomponents—the myeloid and lymphoid lines. Such stem cells are said tobe “totipotent.” Stem cells that are less general but that can stilldifferentiate into several lines are called “pluripotent.” Furtherdifferentiation then occurs among the precursor cells to produce themonocyte, eosinophil, neutrophil, basophil, megakaryocytes, anderythroid lineages from the myeloid line, and T cells, B cells, and NKcells from the lymphoid line. For a background review of the stem cellsee Scientific American 256:86-93 (December 1991).

One of the first breakthroughs into stem cell isolation andidentification came in the late 1980's. In U.S. Pat. No. 4,714,680 (Dec.22, 1987), Civin described a population of pluripotentlympho-hematopoietic cells that were substantially free of maturelymphoid and myeloid cells. Civin also described an antigen, MY-10, anda monoclonal antibody thereto, which was present on those cells. Thosecells made up about 1% of all cells in normal adult bone marrow, andgenerally comprised a mixture of totipotent, and pluripotent stem cellsand lineage committed precursor cells with the latter cellspredominating.

Since that time, MY-10 has been classified by the International Workshopon Human Leukocyte Antigens as falling with the cluster designated as“CD34.” Anti-CD34 monoclonal antibodies are now commercially availablefrom a number of sources including, for example, Becton DickinsonImmunocytometry Systems (“BDIS”).

Anti-CD34 monoclonal antibodies have been used for a number of purposes.Loken, Terstappen and their collaborators have published a series ofpapers describing the maturational stages for various components of thehematopoietic system, such as B lymphocytes (Loken et al., Blood70:1316-1324 (November 1987)), erythroid cells (Loken et al., Blood69:255-263 (January 1987)), and neutrophils (Terstappen et al., Leukemia4:657-663 (September 1990)). The objective of these studies was todefine, starting from the most mature cell and working backwards, thevarious maturational and developmental stages of a lineage committedcell.

Anti-CD34 monoclonal antibodies have also been used to look for earliernon-lineage committed stem cells. For example, Terstappen et al., Blood77:1218-1227 (March 1991), described a subset of human progenitor cellsthat were capable of self-renewal and differentiation into each of thevarious hematopoietic lineages (i.e., a population of cells that includecells that are totipotent). This population was characterized as beingCD34⁺/CD38⁻.

U.S. Pat. No. 5,061,620 to Tsukamoto et al. (Oct. 29, 1991) alsodescribed a population of cells that were capable of self-renewal anddifferentiation. This population of cells was characterized as beingCD34⁺/CD10⁻CD19⁻/CD33⁻ and Thy-1⁺.

Other investigators have attempted to subset CD34⁺ cells from bothperipheral blood and bone marrow. Bender et al., Blood 77:2591-2596(June 1991), used four color flow cytometry with combinations ofmonoclonal antibodies (i.e., anti-CD34, anti-CD33, anti-CD45, anti-CD19,anti-CD7, anti-CD10, anti-CD3, anti-CD20, anti-CD14, anti-CD11b andanti-HLA-DR), to identify and isolate CD34⁺ hematopoietic progenitorcells. Bender et al. were able to identify a number of subsets. Onesubset was CD34⁺/HLA-DR−. This subset had a very small number of cellsand no clear population of this phenotype was resolved. Bender et al.speculated on the ability of this population of cells to give rise toblast cell colonies or cells reconstituting long term cultures basedupon prior work of others.

Sutherland et al., Blood 78:666-672 (August 1991), reported on thedifferential regulation of“primitive” hematopoietic cells in long termculture. They used anti-CD34 and anti-HLA-DR monoclonal antibodies toselect cells that were CD34⁺ and HLA-DR^(dim) or HLA-DR⁻. These cellswere then grown on a unique stromal cell line. The purpose of this workwas to establish a method of long term culture of such cells for thepurposes of studying hematopoiesis and the effect of different growthfactors on hematopoiesis.

Simmons et al., Blood 78:55-62 (July 1991), also reported on the“identification” of a stromal cell precursor in human bone marrow. Usingan antibody they designated “Stro-1,” Simmons et al. were able to removestromal cells from bone marrow. The antigen recognized by this antibodywas not present on colony forming progenitor cells but was present on a“subpopulation of cells experiencing the [CD34] antigen.” Thus, Simmonset al. described the ability of the antibody to separate out stromalcells from hematopoietic cells in bone marrow before culture.

Verfaillie et al., J. Exp. Med. 172:509-520 (August 1990), reported on aCD34⁺/HLA-DR⁺ and CD34⁺/HLA-DR⁻ population of “primitive” progenitorcells. Taking adult marrow, Verfaillie et al. depleted bone marrow oflineage⁺ cells using multiple monoclonal antibodies. Next, fluorescentlylabeled CD34 and HLA-DR monoclonal antibodies were used to selectHLA-DR⁺ and HLA-DR populations that were also CD34⁺. Having isolatedthese two groups, Verfaillie et al. reported that the HLA-DR⁺ cells werebetter in short term culture than the HLA-DR⁻ cells. In long termculture, the reverse was true.

WO 93/25216, published Dec. 23, 1993, teaches a population of humanprimitive stem cells that are capable of self-renewal and that arecapable of differentiating into hematopoietic stem cells and stromalstem cells that give rise to the hematopoietic microenvironment. Thispopulation of cells has the phenotype CD34⁺/CD38/HLA-DR. This populationof cells lacks lineage committed antigens (i.e., is CD33−, CD10−, CD5−,and CD71−). Cells having this phenotype were identified in adult andfetal peripheral blood, bone marrow, thymus, liver, or spleen using acombination of antibodies and selecting for the presence or absence ofthe antigens recognized by these antibodies on the cells. Preferably,the combination of antibodies comprised at least three monoclonalantibodies and more preferably comprised anti-CD34, anti-CD38 andanti-HLA-DR monoclonal antibodies.

WO 94/02157, published Feb. 3, 1994, teaches the isolation of humanhematopoietic stem cells that are CD34⁺, HLA-DR and express the receptorfor the c-kit ligand (KR⁺). This cell population was reportedly usefulfor transplantation and in gene therapy protocols.

To date, the CD34 antigen, as identified by monoclonal antibodies, hasbeen the only known cell surface marker to be used to define thehematopoietic stem cell compartment and has become the marker of choicenot only for the identification of stem cells but also for theirisolation. Published information now indicates the existence ofmonoclonal antibodies that define cell surface markers distinct fromCD34; (i) monoclonal antibody AC133 which binds to a surface protein of96 kDa on approximately 50% of CD34⁺ cells; (ii) monoclonal antibody BB9which binds to a surface protein of 160 kDa on approximately 10-28% ofCD34⁺ cells; and (iii) a non-designated monoclonal antibody that bindsto a glycoprotein 105 on the surface of hematopoietic stem cells.Virtually all of the CFU-S and colony forming unit cells detectable byin vitro stem cell assays express the CD34 antigen. Furthermore, anumber of animal and human studies have demonstrated that purified CD34⁺cells are capable of reconstituting the entire hematopoietic system,suggesting that early engraftment by progenitor cells and long-termmaintenance by primitive stem cells are mediated by this population(See, e.g., Berenson et al., J. Clin. Invest. 81:951-955 (1988)).

The identification and isolation of the most primitive population ofhematopoietic stem cells would be highly advantageous in situationswhere reinfusion of only a small number of long-term repopulating cellswas desired. For example, this would be the case when purging bonemarrow or peripheral blood stem cells of contaminating tumor cells, orwhere genetic manipulation of the stem cells was the objective. CD34expression seems to be stage specific rather than lineage specific withhigher levels of expression seen in primitive progenitors and decreasingexpression levels with cellular maturation (Holyoake & Alcorn, BloodRev. 8(2):113-124 (1994)). Nonetheless, it has never been successfullydemonstrated that stem cells could be purified on the basis of theirCD34 expression levels. The studies described above suggest that CD34⁺cells selected for the absence of lineage specific markers, such as,CD33, CD38, HLA-DR, as well as low Thy-1 labeling (Thy^(lo)) (Craig etal., J. Exp. Med. 177:1331-1342 (1993)), correspond to a stemcell-enriched population. No positive enrichment procedure, however, hasever been described.

Clearly, there is a continuing need in the art to isolate novel markersof primitive stem cell populations so that positively enriched primitivestem cell populations can be obtained and utilized as therapeuticcompositions, as well as in therapeutic methods such as bone marrowtransplantation and gene therapy.

Bone marrow transplantation is an effective therapy for an increasingnumber of diseases. Graft Versus Host Disease (GVHD), however, limitsbone marrow transplantation to recipients with HLA-matched siblingdonors. Even then, approximately half of the allogenic bone marrowtransplantation recipients develop GVHD. Current therapy for GVHD isimperfect and the disease can be disfiguring and/or lethal. Thus, riskof GVHD restricts the use of bone marrow transplantation to patientswith otherwise fatal diseases, such as malignancies, severe aplasticanemia, and congenital immunodeficiency states.

The potential benefits from expanded use of bone marrow transplantationhave stimulated research on the cause and prevention of GVHD. It hasbeen shown that donor T lymphocytes cause GVHD in animals. Removal of Tlymphocytes from donor marrow inocula (“grafts”) prevented thesubsequent development of GVHD in mice, dogs, and monkeys. Similartrials in humans with monoclonal antibodies against human T lymphocytesare now in progress. Preliminary results, however, suggest onlyattenuation of GVHD, not a cure. Similar results have been achieved withE-rosette and soybean lectin depletion of T lymphocytes. Anotherapproach under investigation is the use of anti-T lymphocyte monoclonalantibodies conjugated to toxins, such as ricin.

As of yet, however, GVHD has not been prevented or cured in bone marrowrecipients. Therefore, a continuing need exists for improved methods ofcombating Graft Versus Host Disease.

Donors of bone marrow are also faced with undesirable procedures andrisks. The current procedures for harvesting bone marrow are expensiveand painful. Furthermore, the current donation procedure is accompaniedby the risks associated with anesthesia, analgesia, blood transfusionand possible infection. It would be desirable, therefore, to improve thecurrent method of harvesting hematopoietic stem cells from donors.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns an antibody that recognizes a smallsubset of human hematopoietic mononuclear cells, which may include thehematopoietic stem cell population. An exemplified embodiment of anantibody of the invention is the MG1 monoclonal antibody.

The MG1 antibody recognizes an antigen on a small subset of humanhematopoietic mononuclear cells, but does not bind to antigens onnormal, human mature myeloid cells. The invention also concerns thehybridoma which produce the MG1 antibody.

The present invention also concerns a method for preparing a cellpopulation useful for stem cell transplantation that is positivelyenriched in immature marrow cells and substantially free of maturemyeloid and lymphoid cells.

The present invention also pertains to a method of collecting donationsuseful for stem cell transplantation that avoids the disadvantages ofconventional marrow harvesting techniques.

The present invention also concerns a therapeutic materials and methodsfor transplanting stem cells that can extend the use of stem celltransplantation to the treatment of non-fatal diseases.

The present invention also provides a method of stem cell gene therapy,utilizing antibodies of the present invention.

The present invention also pertains to materials and methods to reduceor eliminate GVHD associated with bone marrow transplantation.

In one embodiment, the present invention provides a method of selectinga population of human cells containing MG1⁺ hematopoietic cellscomprising: (a) providing a cell suspension from human tissue, such asmarrow or blood; (b) contacting said cell suspension with an antibodythat binds the MG1 antigen; and (c) separating and recovering from saidcell suspension the cells bound by said antibody.

In a further embodiment, the present invention provides a method ofselecting a population of human cells containing MG1⁺ hematopoieticcells comprising: (a) providing a cell suspension from human tissue,said tissue selected from the group consisting of marrow and blood; (b)contacting said cell suspension with a solid-phase linked MG1 monoclonalantibody; (c) separating unbound cells from solid-phase linkedmonoclonal antibody after said contacting; and (d) recovering boundcells from said solid-phase linked monoclonal antibody after separatingsaid unbound cells.

Yet another embodiment of the present invention provides a suspension ofhuman cells comprising MG1⁺ hematopoietic cells substantially free ofmature cells, as well as therapeutic methods employing such a cellsuspension.

In a further embodiment, the present invention provides a method of genetherapy utilizing the monoclonal antibody of the present invention toselect for hematopoietic cells that express the MG1 antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

The designation “Miki,” appearing in any of the Figures submittedherein, and described below, is simply the former designation for “MG1,” and thus, should be considered the same as MG1.

FIG. 1 is a flow chart describing the origin of the MG1 hybridoma clone.

FIG. 2 shows an SDS PAGE of purified MG1 IgG. Different volumes ofde-salted, purified MG1 immunoglobulin were analyzed by 10% SDS-PAGE.The gel was fixed, and stained with coomassie blue, and photographed. 2μl, 5 μl, 10 μl and 15 μl indicate the volumes of purified MG1immunoglobulin loaded per well. Marker: BioRad low range prestainedmarkers: the positions of the 106, 80, 49, and 32 kDa bands areindicated.

FIG. 3 shows MG1 antigen expression on the surface of various cellslines. ML-1 (NSC1.1), KG1a, K562, HT 29, and Hel cells were incubatedwith MG1 hybridoma supernatant, then fluorescently labeled with goatanti-mouse FITC-labeled polyclonal antibody, and examined for thepresence of surface labeling by flow cytometry.

FIG. 4 shows a flow cytometry analysis of whole human bone marrow usingfluorescein-labeled MG1 monoclonal antibody. Whole normal adult humanbone marrow was labeled with MG1-FITC and anti-CD34-PE (QBend 10),fixed, and analyzed by flow cytometry. 50,000 events were collected. Thex-axis depicts MG1-associated fluorescence intensity. The y-axis depictsCD34-associated fluorescence intensity. MG1 positive cells (lower rightquadrant) represent less than 1% of all bone marrow cells, whereas,CD34⁺ cells (upper left quadrant) represent approximately 1% of all bonemarrow cells. FIG. 4 shows that MG1 recognizes only a very smallpopulation (less than 1%) of cells within bone marrow, and that lessthan 3% of CD34+ cells co-express MG1 antigen, suggesting a very smalloverlap of the two populations. Quad, quadrant; UL, upper left; LR,lower right.

FIG. 5 shows hybridization of Western blot with MG1 antibody showing theapproximate molecular weight of the MG1 antigen. Cell extracts from 10⁶cells, prepared from various cell lines, were separated by SDS PAGEthrough 10% gels, and transferred to nitrocellulose membranes. Themembrane was probed with MG1 primary antibody (hybridoma supernatant ata 1:100 dilution), and HRP-conjugated sheep anti-mouse IgG secondaryantibody, and visualized by ECL. Marker: BioRad high range prestainedmarkers; the positions of the 205, 116, and 80 kDa bands are indicatedwith short lines. ML-1; NSC1.1 cell extracts. HT-29, HEL, K562, andKG1a, refer to cell extracts from the respective cell lines.

FIG. 6 shows hybridization of Western blot with MG1 hybridomasupernatant. Cell extracts from 10⁶ cells, prepared from various celllines, were separated by SDS PAGE through 10% gels, and transferred tonitrocellulose membranes. The membrane was hybridized to MG1 primaryantibody (hybridoma supernatant at a 1:10 dilution), and HRP-conjugatedsheep anti-mouse IgG secondary antibody, and visualized by ECL. Marker:BioRad low range prestained markers: the positions of the 106, and 80kDa bands are indicated with hyphens. ML-1; NSC1.1 cell extracts. HT-29,HEL, K562, and KG1a, refer to cell extracts from the respective celllines.

FIG. 7 shows hybridization of Western blot with MG1 purified antibody.Comparison with FIG. 6 shows that the purified antibody and hybridomasupernatant recognize the same protein in cell extracts. Cell extractsfrom 10⁶ cells, prepared from various cell lines, were separated by SDSPAGE through 10% gels, and transferred to nitrocellulose membranes. Themembrane was hybridized to MG1 primary antibody (purified IgG), andHRP-conjugated sheep anti-mouse IgG secondary antibody, and visualizedby ECL. Marker: BioRad low range prestained markers: the positions ofthe 106, and 80 kDa bands are indicated with hyphens. ML-1; NSC1.1 cellextracts. HT-29, HEL, K562, and KG1a, refer to cell extracts from therespective cell lines.

FIG. 8 shows hybridization of blood cell extracts with MG1 antibody.Cell extracts from NSC1.1 cells or from different populations of bloodcells separated over PERCOLL gradients, were separated by SDS PAGEthrough 10% gels, and transferred to nitrocellulose membranes. Themembrane was probed with purified MG1 IgG as primary antibody, andHRP-conjugated sheep anti-mouse IgG secondary antibody, and visualizedby ECL. ML-1 lysate; lysate from 10⁶ NSC1.1 cells. RBCs, Granulocytes,Lymphocytes, Thrombocytes; lysates prepared from 10⁵ red blood cells,granulocytes, lymphocytes and thrombocytes, respectively. Marker: BioRadhigh range prestained markers; the positions of the 205, 116, and 80 kDabands are indicated as short lines.

FIG. 9 shows a Western blot demonstrating that the MG1 antigen isglycosylated. 100 ml crude NSC1.1 lysate was incubated with theindicated glycosidases at 37° C. overnight. The samples were thendiluted and separated by SDS PAGE through 10% gels, and transferred tonitrocellulose membranes. The membranes were hybridized to MG1 primaryantibody, and HRP-conjugated sheep anti-mouse IgG secondary antibody,and visualized by ECL. Marker: BioRad high range prestained markers; thepositions of the 205 and 116 kDa bands are indicated. Control; lysatesincubated without enzyme. O-glycosidase; lysates incubated with 2.5 mUof O-glycosidase. N-glycosidase; lysates incubated with 6 unitsN-glycosidase. O+N-glycosidase; lysates incubated with both O- andN-glycosidases.

FIG. 10 shows partial purification of the MG1 antigen by affinity columnchromatography. Lysates prepared from NSC1.1 cells were loaded onto animmunoaffinity column containing bound purified MG1 monoclonal antibody.The column was washed, and the bound protein was eluted with 0.1 Mglycine (pH 2.7) into 1.0 M Tris (pH 9.0). This eluate was concentratedby ultrafiltration, and examined by SDS PAGE. The size of the MG1antigen, as calculated by a regression analysis of the two proteinmarker lanes (non-prestained), is 186 kDa±5%. Legend: pm, prestainedprotein molecular weight markers; m, Novex ‘Mark 12’ protein molecularweight markers (The positions of the 200, 116.3, 97.4, 66.3, 55.4, 36.5,and 31.0 kDa bands are indicated); lys 1, aliquot of the NSC 1.1 lysate,and lys 2, aliquot of a second protein extraction of the NSC1.1lysate;f-t, the flow-through from loading the affinity column; elu,eluate; conc. elu, concentrated eluate; re-elu, second elution from theaffinity column.

FIG. 11 shows a Western blot analysis for detection of lamininreactivity using NSC1.1 lysate and a polyclonal antibody against humanlaminin. NSC1.1 lysates (10 μl per lane), NSC1.1 conditioned medium (10μl of condition medium concentrated 10× on a Centriprep column) andpurified mouse laminin (approximately 21 μg per lane) were separated onSDS-PAGE through 10% gels and transferred to nitrocellulose membranes.The membrane was separated into three parts, and the individual partshybridized to anti-laminin polyclonal antibody (1:2000 dilution), or MG1monoclonal antibody (1/10 dilution), or PBS alone. Following incubationwith the primary antibody, the membranes were washed, and horseradishperoxidase labeled goat anti-mouse (or donkey anti-rabbit) IgG secondaryantibody was added (1/2000 dilution). Detection of antigen/antibodycomplexes was performed using the chemiluminescent reagent fromAmersham's ECL system. The resulting X-ray films were developed using aKodak M35A X-OMAT processor and the autoradiographs examined forpositive reactions. The positions of the 205, 116, and 80 kDa bands ofthe BioRad high range prestained marker are indicated with short lines.Legend: ML-1 lysate, NSC1.1 lysates; ML-1 CM, NSC1.1 conditioned medium;Miki, 1,2,3: lanes labeled with MG1 monoclonal primary antibody; PBS,1,2,3: no primary antibody; polyclonal laminin Ab, 1,2,3: lanes labeledwith anti-laminin polyclonal antibody.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 shows a partial amino acid sequence of the MG1 antigenpolypeptide according to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns a novel polypeptide antigen. The antigen,which is referred to herein as the “MG1 antigen,” or alternatively, the“MG1 ligand,” is expressed on a very small population of humanhematopoietic cells, which may include pluripotent hematopoietic stemcells. Stem cells have the ability to restore, when transplanted, theproduction of myeloid and lymphoid cells to a patient who has lost suchproduction due to, for example, radiation therapy or disease. Theantigen disclosed herein is not expressed by mature myeloid cells. Thenewly discovered antigen may help to define a population ofhematopoietic cells desirable for use in a bone marrow transplant. Thisantigen was identified by a monoclonal antibody raised against the humanNSC1.1 cell line, which is derived from the ML-1 cell line.

The present invention also concerns antibody that can bind to the MG1antigen. An exemplified embodiment of the invention is a monoclonalantibody, referred to herein as “MG1,” that facilitates the isolation ofthe desired cells and makes possible improved therapeutic techniquesthat significantly contribute to the understanding and prevention ofGraft Versus Host Disease. The isolated stem cells can also be employedto produce panels of monoclonal antibodies to stem cells. The monoclonalantibodies of the invention can also be employed in stem cell genetherapy.

The ML-1 cell line used as an immunogen as described herein was derivedfrom an enriched, immunoselected, CD34⁺ population isolated from normalhuman cadaveric bone marrow (U.S. Pat. No. 5,650,299, issued Jul. 22,1997). The immunoselected CD34⁺ cells were cultured, in vitro, in thepresence of either recombinant IL-3, and IL-6, or native, semi-purifiedstem cell proliferation factor (SCPF). SCPF is a cytokine that promotesthe proliferation of primitive hematopoietic stem cells whilemaintaining their CD34 phenotype. The cells were grown in the presenceof these exogenous cytokines for a period of several months. CD34 cellsgrown in the presence of IL-3 and IL-6 differentiated and the cultureswere lost. However, the culture supplemented with SCPF wereSCPF-dependent and continued to proliferate. The SCPF-dependency waslost over time and the cell cultures became SCPF-independent. Thispopulation of cells was examined by flow cytometry and was shown toexpress CD34. This cell line was designated ML-1. The ML-1 cell line wascloned by limiting dilution and rescreened by flow cytometry and a subclone of ML-1 was established that was phenotypically more primitivethan that of the parental cell line. This cell line was designatedNSC1.1. Given the primitive nature of this cell population, this cloneof ML-1 was used as the immunogen for the hybridoma program.

The MG1-ligand is expressed as a cell-surface antigen on the NSC1.1 cellline. The antigen can be immunoprecipitated from extracts of this cellline as a glycosylated protein of approximately 186±5% kD (kilodalton)apparent molecular weight.

Antibodies that specifically label a subset of hematopoietic progenitorsare extremely useful in hematopoietic research because they allow theisolation of relatively pure populations of immature hematopoietic cellsin a single step. Cells recovered with MG1 antibody could be anappropriate normal cell population to compare with leukemic blast cellsand to use in studies on the mechanisms of action of cells, factors, andgenes that regulate hematopoietic cell proliferation anddifferentiation.

MG1 antibody did not recognize cells from three human leukemic celllines (e.g., KG-1a, K562, HEL 92.1.7), and did not bind to humanperipheral blood cells. MG1 did not bind to mouse bone marrow cells.

Monoclonal anti-stem cell antibodies can be produced readily by oneskilled in the art. The general methodology for making monoclonalantibodies using hybridoma technology is now well known in the art. See,e.g., M. Schreier et al., Hybridoma Techniques (Cold Spring HarborLaboratory 1980); Hammerling et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier Biomedical Press 1981); Kennett et al., MonoclonalAntibodies (Plenum Press 1980). Immortal, antibody-secreting cell linescan also be produced by techniques other than fusion, such as directtransformation of B-lymphocytes with oncogenic DNA or EBV. Severalantigen sources can be used, if desired, to challenge the normalB-lymphocyte population that is later converted to an immortal cellline.

For example, the NSC1.1 cell line can be used as an immunogen tochallenge the mammal (e.g., mouse, rat, hamster, etc.) used as a sourcefor normal B-lymphocytes. The antigen-stimulated B-lymphocytes are thenharvested and fused to an immortal cell line or transformed into animmortal cell line by any appropriate technique. A preferred hybridomaproducing the monoclonal MG1 antibody is produced by challenging a mousewith the NSC1.1 cell line and fusing the recovered B-lymphocytes with animmortal SP2/0-Ag14 myeloma cell. Antibody-producing immortal cells canbe screened for anti-stem cell antibody production by selecting clonesthat are strongly reactive with the NSC1.1 cells, but not reactive withgranulocytes from a panel of human donors. Antibodies produced by cloneswhich show those properties can then be screened for the additionalproperties of anti-stem cell antibodies.

A mouse hybridoma producing monoclonal MG1 antibody was deposited withthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, on Nov. 6, 1996, and assigned ATCC AccessionNo. HB12232.

The subject cell line has been deposited under conditions that assurethat access to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 USC122. Thedeposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject cell line deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least 30 (thirty)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the cell line. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

In a preferred embodiment, the present invention encompasses anymonoclonal antibody that recognizes the MG1 antigen, i.e., the antigenrecognized by antibody from the hybridoma ATCC HB12232. In anotherpreferred embodiment, the present invention contemplates monoclonalantibodies that correspond to the monoclonal antibody produced by ATCCHB12232, and, in a particularly preferred embodiment, the ATCC HB12232antibody. One antibody corresponds to another antibody if they bothrecognize the same or overlapping antigen binding sites as demonstratedby, for example, a binding inhibition assay.

An alternative to the above method of producing monoclonal antibodiesemploys the MG1 antigen directly as an immunogen. The monoclonalantibody produced by hybridoma ATCC HB12232 can be readily employed topurify the MG1 antigen. In one example of immunopurification, MG1antigen can be immunoprecipitated from cell extracts of the NSC1.1 cellline. The precipitated antigen can be used as an immunogen in place ofthe NSC1.1 cell line in the above method. By application of any of theabove methods, one skilled in the art can readily produce a panel ofmonoclonal antibodies, screen them with partially purified MG1, andobtain anti-MG1-ligand antibodies.

Another alternative is to use an MG1 antibody in the production ofmonoclonal antibodies that recognize different antigens on MG1⁺ cells.The cells isolated from blood and marrow with MG1 antibody can be usedas an immunogen, as described above, to produce a panel of monoclonalantibodies against MG1⁺ cells. The production of such antibodies isgreatly facilitated by the use of substantially pure populations ofhematopoietic cells provided by the MG1 antibody. The specificities ofsuch antibodies can be determined readily through routine screening byone skilled in the art. Thus, additional stage-specific, lineageindependent antigens (and antibodies to these antigens) can beidentified by those skilled in the art.

MG1 recognizes a marker on the surface of hematopoietic cells that isdistinct from CD34. Consequently, use of the MG1 antibody provides analternative (or additional) means for the positive selection of a subsetof this population from human bone marrow or peripheral blood. Giventhat MG1⁺ cell populations represent only a subset of the CD34population, antibodies immunoreactive with the MG1 antigen can be usedin the field of tumor cell purging of bone marrow or peripheral bloodstem cells, as well as in the preparation of stem cell populations forgenetic therapy.

The antibodies according to the subject invention may be eithermonoclonal, polyclonal, or a mixture of monoclonal and/or polyclonalantibodies. The antibody may comprise whole antibody or antigen-bindingfragments thereof, such as Fab₂, Fab and Fv fragments. Antigen bindingfragments can be prepared using conventional techniques known in theart, such as proteolytic digestion of antibody by papain or pepsin, orthrough standard genetic engineering techniques known in the art.Monoclonal antibodies exemplified herein can be engineered so as tochange the isotype of the antibody. For example, the MG1 antibody, whichis an IgG_(2A) isotype, can be engineered as an IgG₁, IgG_(2B), or otherisotypes. Also contemplated by the subject invention are antibodies thatare reactive with the MG1 antibody and which have been engineered tocomprise human antibody constant regions. “Humanized” antibodies can beprepared using standard methods known in the art. See, for example, U.S.Pat. No. 5,585,089 (issued Dec. 17, 1996), the disclosure of which ishereby incorporated by reference.

The antibodies of the subject invention can be labeled according tostandard methods known in the art. For example, antibodies can belabeled with detectable labels such as fluorescein, rhodamine andradioactive isotopes.

As indicated above, one application for monoclonal antibodies to lineageindependent antigens on stem cells is the isolation of a highly enrichedsource of stem cells for human bone marrow transplantation. Such sourcesof stem cells can prevent or attenuate Graft Versus Host Disease.Anti-stem cell monoclonal antibodies can also be used to isolate stemcells for autologous reinfusion, for example, in the treatment ofantigen-negative leukemias or other malignancies.

The present invention contemplates the use of any method employing theMG1 monoclonal antibody to separate stem cells from mature lymphocytesin the marrow or blood. Generally, a cell suspension prepared from humantissue containing cells (i.e., marrow or blood cells) is brought intocontact with the MG1 monoclonal antibody. Cells that have been bound bythe monoclonal antibody are then separated from unbound cells by anymeans known to those skilled in the art.

Various methods of separating antibody-bound cells from unbound cellsare known. For example, the antibody bound to the cell (or ananti-isotype antibody) can be labeled and then the cells separated by amechanical cell sorter that detects the presence of the label.Fluorescence-activated cell sorters are well known in the art. In onepreferred embodiment, the anti-stem cell antibody is attached to a solidsupport. Various solid supports are known to those of skill in the art,including, but not limited to, agarose beads, polystyrene beads, hollowfiber membranes, polymers, and plastic petri dishes. Cells that arebound by the antibody can be removed from the cell suspension by simplyphysically separating the solid support from the cell suspension.Preferred protocols, however, will be described.

Selective cytophoresis can be used to produce a cell suspension fromhuman bone marrow or blood containing pluripotent hematopoietic stemcells. For example, marrow can be harvested from a donor (the patient inthe case of an autologous transplant; a donor in the case of anallogeneic transplant) by any appropriate means. The marrow can beprocessed as desired, depending mainly upon the use intended for therecovered cells. The suspension of marrow cells is allowed to physicallycontact, for example, a solid phase-linked monoclonal antibody thatrecognizes an antigen on the desired cells. The solid phase-linking cancomprise, for instance, adsorbing the antibodies to a plastic,nitrocellulose, or other surface. The antibodies can also be adsorbed onto the walls of the large pores (sufficiently large to permitflow-through of cells) of a hollow fiber membrane. Alternatively, theantibodies can be covalently linked to a surface or bead, such asPharmacia Sepharose 6 MB macrobeads. The exact conditions and durationof incubation for the solid phase-linked antibodies with the marrow cellsuspension will depend upon several factors specific to the systememployed. The selection of appropriate conditions, however, is wellwithin the skill of the art.

The unbound cells are then eluted or washed away with physiologic bufferafter allowing sufficient time for the stem cells to be bound. Theunbound marrow cells can be recovered and used for other purposes ordiscarded after appropriate testing has been done to ensure that thedesired separation had been achieved. The bound cells are then separatedfrom the solid phase by any appropriate method, depending mainly uponthe nature of the solid phase and the antibody. For example, bound cellscan be eluted from a plastic petri dish by vigorous agitation.Alternatively, bound cells can be eluted by enzymatically “nicking” ordigesting a enzyme-sensitive “spacer” sequence between the solid phaseand the antibody. Spacers bound to agarose beads are commerciallyavailable from, for example, Pharmacia.

The eluted, enriched fraction of cells may then be washed with a bufferby centrifugation and either cryopreserved in a viable state for lateruse according to conventional technology or immediately infusedintravenously into the transplant recipient.

In a particularly preferred embodiment, stem cells can be recovereddirectly from blood using essentially the above methodology. Forexample, blood can be withdrawn directly from the circulatory system ofa donor and percolated continuously through a device (e.g., a column)containing the solid phase-linked monoclonal antibody to stem cells andthe stem cell-depleted blood can be returned immediately to the donor'scirculatory system using, for example, a conventional hemapheresismachine. When a sufficient volume of blood has been processed to allowthe desired number of stem cells to bind to the column, the patient isdisconnected from the machine. Such a method is extremely desirablebecause it allows rare peripheral blood stem cells to be harvested froma very large volume of blood, sparing the donor the expense and pain ofharvesting bone marrow and the associated risks of anesthesia,analgesia, blood transfusion, and infection.

The above cell populations containing MG1⁺ enriched human hematopoieticcells can be used in therapeutic methods such as stem celltransplantation, as well as other methods that are readily apparent tothose skilled in the art. For example, such cell populations can beintravenously administered to a patient requiring a bone marrowtransplant in an amount sufficient to reconstitute the patient'shematopoietic and immune system. Precise, effective quantities can bereadily determined by those skilled in the art and will depend, ofcourse, upon the exact condition being treated by the therapy. In manyapplications, however, an amount containing approximately the samenumber of stem cells found in one-half to one liter of aspirated marrowshould be adequate.

In another embodiment, the MG1 monoclonal antibody can be used toisolate MG⁺ cells, which can be used in various protocols of genetictherapy.

The optimal choice of target tissue for gene therapy is a long-lived,preferably self-renewing cell. The hematopoietic stem cell isparticularly attractive as a target for gene therapy for severalreasons. First, the procedures for the collection, cryopreservation, andreinfusion of human bone marrow are well developed, and the efficacywell established. Second, the use of stem cells or very earlypluripotential precursor cells would assure long term maintenance of thegenetically modified cells, and thus reduce the number of interventionsrequired. Third, one of the obstacles faced by gene therapists is thatsustained high level expression of transgenes has been difficult toachieve in large outbred mammals (Blaese, R. M., Clinical Immunology andImmunopathology, (61):547-555 (1991); Miller, A. D., Nature 357:455-460(1992)). One of the ways to address this problem has been to useexpression systems adapted to the target tissue (Kay et al., Hum. GeneTher. 3:641-647 (1992)). In the hematopoietic system, precursor (stem)cells can differentiate along one of three developmental pathways thatproduce large numbers of terminally differentiated cells, myeloid,lymphoid and erythroid cells. The control of gene expression during thedevelopment of the hematopoietic system has been extensively studied(Evans et al., Ann. Rev. Cell Biol. 6:95-124 (1990)), and elementsimplicated in the tissue-specific expression of genes have beenidentified for all three developmental pathways. The therapeutictransgene could be genetically modified to be constitutively expressedor expressed specifically in one of the differentiated hematopoieticlineages. Fourth, small numbers of hematopoietic stem cells produce verylarge numbers of differentiated cells; this diminishes the burden on thetransducing procedure to be of very high efficiency or throughput sincea small population of genetically modified stem cells will generate alarge population of genetically modified cells within the patient.Finally, since small numbers of genetically modified cells arenecessary, the risk associated with the introduction of large numbers ofgenetically modified cells into patients is also diminished.

The use of a stem cell-specific antibody need not be limited to thepurification of stem cells prior to a transfection procedure. With thegoal of generating vectors for in vivo gene therapy, it has beenproposed to engineer into the gene therapy vectors themselves,mechanisms by which the vector will recognize its target cell (andpreferably only its target) within the context of the entire organism.See, Kasahara et al., Science 266:1373-1376 (1994); Michael & Curiel,Gene Therapy 1:223-232 (1994); Chatterjee et al., Ann. N.Y. Acad. Sci.770:79-90 (1995); Schwarzenberger et al., Blood 87:472-478 (1996). Byincorporating stem cell-specific antibodies into a vector, it may bepossible to generate vectors that will recognize and targethematopoietic stem cells in the patient's bone marrow. Specifically, theantibody could be incorporated into liposome vectors, (Hughes et al.,Cancer Res. 49:6214-6220 (1989); Wang & Huang, Biochemistry 28:9508-9514(1989); Ahmad et al., Cancer Res. 53:1484-1488 (1993)), poly-L lysineconjugate vectors (Michael & Curiel, supra; Schwarzenberger et al.,supra), or into viral vectors, including but not limited to adenoviralvectors, retroviral vectors, (Russell et al., Nucleic Acids Res.21:1081-1085 (1993); Somia et al., Proc. Natl. Acad. Sci. USA92:7570-7574 (1995)), and adeno-associated vectors (Chatterjee et al.,supra), modified to express on the vector surface, the antibody itselfor proteins which would bind the antibody to the vector surface (such asthe Fc receptor).

These vectors, although partly conceived for use in in vivo gene therapycan also be used to target the same MG1⁺ cells in ex vivo applications,on either pre-selected MG1⁺ cells, or on whole bone marrow, mobilizedperipheral blood stem cells, or cord blood stem cells.

In a preferred method of in vivo gene therapy, genetic disorders, suchas, e.g., sickle cell anemia, β-thalassemia, Fanconi anemia, and otherhemoglobinopathies, maybe corrected by the introduction of the normalgene into a human stem cell, which can then be transplanted into apatient's bone marrow. The treatment of genetic disease usinggenetically modified stem cells is not limited to the treatment ofhematopoietic tissues and diseases, but can also be extended to diseasesin which the presence of a circulating protein is of clinical benefit,such as Gaucher disease and hemophilia A and B.

To effect gene therapy with a substantially pure population of humanprogenitor cells, the following method may be used to insert a gene intothese cells. For a general review of the methodologies, see Friedmann,T., Science 244:1275-1281 (June 1989) and Lancet 1: 1271-1272 (Jun. 4,1988).

A therapeutic gene can be introduced into the population of purifiedstem cells, isolated as above by; (1) physical methods such ascoprecipitation with calcium phosphate, electroporation ormicroinjection (e.g., U.S. Pat. No. 4,873,191), and/or (2) the use ofviral vectors such as adenoviral, or retroviral vectors. In the lattercase, the DNA of the retrovirus is cut with a restriction enzyme and thehuman DNA containing the desired sequence is inserted and ligated. Theretrovirus containing the insertion is then infected into the stemcells. The stem cells can then be assayed for production of the desiredprotein. See, e.g., U.S. Pat. No. 4,902,783.

In general, molecular DNA cloning methods are well known in the art andare not limiting in the practice of this invention. For a furtherdescription of similar methods, see Friedmann, T., Science 244:1275-1281(1989) and Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrooket al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989).

To transplant the stem cells containing the desired gene, the cells maybe introduced into the bone marrow of the patient by conventional meansof bone marrow transfer. Typically, this involves the intravenousdelivery of the cells over a period of time. The bone marrow of thepatient may be lethally irradiated prior to infusion to assure that thetransplanted stem cells fully replace the existing bone marrow cells.See U.S. Pat. No. 4,721,096 to Naughton et al. (Jan. 26, 1988).

In another method of gene therapy, drug resistance genes (i.e.,dihydrofolate reductase (dhfr), MDR1) can be introduced into normal stemcells and then transplanted into a cancer patient undergoingchemotherapy to protect the healthy stem cells from the toxic effects ofthe drug. This procedure would allow more aggressive chemotherapeuticregimens to be employed. (Guigon et al., Bone Marrow Transplant 13:93-95(1994); Baum et al., J. Virol. 69:7541-7547(1995)). MG1⁺ cellpopulations maybe useful in this method. Modified stem cells also haveapplications in the treatment of certain acquired diseases such as HIVinfections (Nienhuis et al., Cancer 67(10 Suppl.):2700-2704 (1991);Bahner et al., J. Virol. 70:4352-4360 (1996)).

In another method of gene therapy, the MG1 antibody can be used in anantibody modified delivery system to target drug delivery specificallyto MG1⁺ hematopoietic cells in vivo. See, e.g., Ahmad et al., CancerRes. 53:1484-1488 (1993).

The subject invention also pertains to the gene encoding the MG1 antigenpolypeptide. Contemplated within the scope of the invention are naturaland allelic variant polynucleotide sequences encoding the MG1 antigen,as well as degenerate polynucleotide sequences that encode the MG1antigen polypeptide. Polynucleotide sequences encoding the MG1 antigencan be readily obtained using materials of the subject invention andstandard methods known in the art. For example, a cDNA library can beprepared from NSC1.1 cells and that cDNA library inserted into anappropriate expression system, such as lambda phage. Clones can then bescreened for expression products using, for example, an MG1 antibody.Clones that are positive for expression of a polypeptide that binds toMG1 antibody can be further evaluated by sequencing of the cDNA insert,Western blot, etc.

Polynucleotide sequences encoding the MG1 antigen can also be obtainedusing degenerate oligonucleotide probes based on the amino acid sequenceof the MG1 antigen in conjunction with standard RACE procedures known inthe art. For example, 5′ RACE can be performed using a MARATHON cDNAamplification kit (CLONTECH Laboratories, Palo Alto, Calif.) to amplifya 5′ RACE fragment from polyA⁺ RNA obtained from cells that express theMG1 antigen. The RACE fragments generated using the degenerateoligonucleotide probes can then be cloned and characterized bysequencing. Full length cDNA sequences can then be generated by end toend PCR or by conventional cloning methods known in the art.

Other methods for screening for polynucleotide sequences encoding theMG1 antigen are known in the art. These include, for example, screeningDNA libraries using degenerate oligonucleotide probes that can beprepared based on the partial amino acid sequence of the MG1 antigen.Also contemplated within the scope of the present invention arefragments and variants of the polynucleotide encoding MG1 antigen, aswell as fragments and variants of the MG1 antigen itself. The fragmentsof the subject polynucleotides can be readily prepared using standardmethods known in the art. For example, digestion using the BAL31exonuclease can be used to prepare 5′ and 3′ nucleotide deletions.

The subject invention also pertains to anti-idiotypic antibodies thatpossess binding specificity for idiotypic determinants associated withanti-MG1 antigen antibodies of the present invention. The anti-idiotypicantibodies of the invention can be prepared using the anti-MG1 antigenantibodies as an immunogen according to standard methods for producinganti-idiotypic antibodies known in the art. Also included within thescope of the present invention are antigen binding fragments of wholeanti-idiotypic antibody, wherein the fragments retain substantial by thesame binding specificity as the whole antibody molecule. The antigenbinding fragments include, for example, Fab₂, Fab and Fv fragments.

The subject invention also concerns kits comprising a compartmentcontaining at least one anti-MG1 antigen antibody, anti-idiotypicantibody or an MG1 antigen. In one embodiment, the anti-MG1 antigenantibody is the MG1 antibody disclosed herein.

Antibodies to the MG1 antigen can also be used to investigatedifferential expression of the MG1 antigen on tumor cells versus normalcells. Thus, the subject invention also concerns methods for identifyingtumor cells, such as leukemic cells, from normal cells by contacting asample with an antibody of the invention and determining whether theantibody binds to any of the test cells. In a preferred embodiment, MG1antigen expression on cells is determined using the MG1 antibody of thepresent invention.

The subject invention also concerns methods for treating tumors inpatients using an antibody of the subject invention that binds to an MG1antigen. In a preferred embodiment, the method comprises administeringan effective amount of an MG1 antigen binding antibody to a patient inneed of such treatment. Methods for administering antibodies to treatvarious disease states are known in the art. Preferably, the antibody ismodified in a manner so as to minimize any immune response to theantibody when it is administered to the patient. For example, theantibody can be “humanized,” such as by replacing non-human constantregions of the antibody with human constant regions, according tomethods known in the art. A variety of toxic agents capable of killingor inhibiting the replication of a cell can be conjugated to theantibody. For example, a variety of cytotoxic agents are available inthe art. These include, for example, radionuclides (Iodine-131,Yttrium-90 and the like), chemotherapeutic agents (such as methotrexate,cisplatinum and the like) and cytotoxic proteins (such as ricin,exotoxins, diphtheria toxins and the like). In one embodiment, themethod can be used to treat leukemia.

The following examples are provided to illustrate specific embodimentsof the present invention. The examples are included for illustrativepurposes only, and are not intended to limit the scope of the presentinvention.

MATERIALS AND METHODS

Preparation of Cell Line for Use as an Immunogen

NSC1.1 cells were grown in T-75 cm² tissue culture flasks using thefollowing growth medium: Iscove's modified Dulbecco's medium (IMDM)supplemented with 10% fetal bovine serum (FBS), L glutamine (0.292mg/ml) and antibiotics (50 Units/ml penicillin, and 50 Units/mlstreptomycin). Once the NSC1.1 cells reached confluency, thesupernatants were harvested, centrifuged (2000 rpm for 4 minutes at roomtemp.), and the cell pellet resuspended in IMDM growth medium (as statedabove). The concentration of viable cells at time of harvest wasestimated (using trypan blue dye exclusion) and the cell suspensionadjusted to a concentration of 1.5×10⁸ cells/ml. The cells were thenwashed four times in sterile phosphate buffered saline (PBS) at pH 7.5and finally resuspended in one ml of PBS at a concentration of1.5×10⁸/ml.

Mice Immunization Protocol

Three 23-27 day-old female Balb/c mice (Charles River Laboratories, MD)were injected with 200 μl of the NSC1.1 cell suspension (3×10⁷ totalcells) via the intra-peritoneal (I/P) route. As a control, one mouse wasinjected, I/P, with 200 μl of PBS only. Following the initial injection,the mice were immunized in the manner and over the time course set outbelow:

Time Protocol Route Inoculum Day 0 (Jan. 23, 1995) Primary Immun. I/P 3× 10⁷ Ml-1 cells Day 15 (Feb. 7, 1995) First Booster I/P 3 × 10⁷ Ml-1cells Day 23 (Feb. 17, 1995) Second Booster I/P 3 × 10⁷ Ml-1 cells Day33 (Feb. 27, 1995 Final Booster^(i)  I/V^(ii)    10⁷ Ml-1 cells Day 36(Mar. 2, 1996) Harvest spleen for fusion & hybridoma production Notes:^((i))Blood was collected (tail vein bleed) from all experimental miceon day 33. The sera from these bleeds was removed and the level of theanti NSC1.1 response was measured by both flow cytometry andimmunocytochemistry (see attached methods). ^((ii))I/V = IntravenousinjectionEstablished Cell Lines Used

Sp2/0-Ag14: Sp2/0-Ag14 (ATCC CRL-1581) is anon-secreting myeloma hybridof murine origin that is routinely used in the fusion process ofhybridoma production.

KG1a: KG1a (ATCC CCL-246.1) is a variant subline of the human, acutemyelogenous leukemia cell line KG1 (ATCC CCL-246). KG1a was the cellline that was used to manufacture the MY10 monoclonal antibody thatdefines the CD34 antigen. See U.S. Pat. No. 4,965,204 (Oct. 25, 1990) toCivin.

K562: K562 (ATCC CCL-243) is a continuous cell line established from ahuman with chronic myelogenous leukemia. The cell line is characterizedas a highly undifferentiated blast of the granulocytic series. Recentstudies indicate that the K562 cells are undifferentiated blasts thatare multipotential and capable of differentiating into progenitors ofthe erythrocytic, granulocytic, and monocytic series.

HT-29: HT-29 (ATCC HTB-38) is a human colon adenocarcinoma.

HEL 92.1.7: Hel 92.1.7 (ATCC TIB-180) is a lymphoblastic-like cell linederived from an erythroleukemia. This cell line is capable of bothspontaneous and induced globin synthesis.

Hybridoma Production

Prior to the fusion, Sp2/0-Ag14 myeloma cells were tested for theirsensitivity to Hypoxanthine, Aminopterin, Thymidine (HAT) containingmedium. The Sp2/0-Ag14 myeloma cells are hypoxanthine-guaninephosphoribosyl-transferase (HGPRT) negative and, therefore, aresensitive to the presence of HAT whereas normal spleen cells are HGPRT⁺and are resistant to HAT. The HGPRT mutation was, therefore, used in thepositive selection of hybrid cells (spleen/myeloma).

HAT-sensitive Sp2/0-Ag14 cells were harvested, from culture, centrifuged(2000 rpm, 4 minutes), and the pellets resuspended in IMDM supplementedwith L glutamine (0.292 mg/ml) and antibiotics (50 Units/ml penicillin,and 50 Units/ml streptomycin). Cell counts were performed by trypan bluedye exclusion.

The 3 experimental mice (immunized with NSC1.1 cells as described above)were sacrificed by cervical dislocation and their spleens removed usingaseptic techniques. The spleens were kept separate and placed in 100 mmpetri dishes containing 10 ml of the IMDM supplemented with L glutamine(0.292 mg/ml) and antibiotics (50 Units/ml penicillin, and 50 Units/mlstreptomycin). Cell suspensions from each spleen were made in the abovemedium, washed twice and viable cells enumerated, by trypan blue dyeexclusion. The Sp2/0-Ag14 myeloma cell and spleen cell suspension weremixed together at a ratio of 1:5 (myeloma:spleen) in the presence ofpolyethylene glycol (PEG) 1500 (Boehringer Mannheim).

The fusion mixture was then incubated at 37° C. for 10 minutes.Following the incubation step, the cell-fusion mixture was centrifugedand resuspended in 30 ml of fresh IMDM supplemented with 20% fetalbovine serum (FBS), L glutamine (0.292 mg/ml) and antibiotics (50Units/ml penicillin, and 50 Units/ml streptomycin). The myeloma/spleencell fusion mixture, for each mouse, was added in 100 μl volumes (5×10⁵spleen cells/well) to each well of three 96-well microplates. Themicroplates (a total of 9 plates) were then incubated at 37° C. in ahumidified atmosphere of 5% CO₂ in air. Four hours after incubation at37° C., 100 μl of IMDM containing 20% fetal bovine serum (FBS), Lglutamine (0.292 mg/ml) and antibiotics (50 Units/ml penicillin, and 50Units/ml streptomycin) and further supplemented with 2×HAT was added toeach well of the nine 96-well microplates.

All the microplates were then re-incubated at 37° C. in a humidifiedatmosphere of 5% CO₂ in air and checked daily for the appearance ofhybridoma growth. Every third day, 100 μl of supernatant from each ofthe wells was removed and replenished with fresh IMDM growth media plusHAT. The media removed from the individual wells was then used to screenfor positive NSC1.1 reactivity. Screening was carried out using asandwich ELISA and/or immunocytochemistry.

The multi-clone wells that were identified as having NSC1.1 specificimmunoglobulins in the supernatant, by either test, were grown to highcell density and subsequently passaged into new wells of a 96-wellmicroplate. These multi-clones were routinely screened for reactivitywith the immunizing NSC1.1 cell line. If the clone(s) maintained theirreactivity they were expanded to tissue culture containers with greatersurface area. Excess cells from each positive clone were stored inliquid nitrogen. Once positive clones were established, the antibodyreactivity was tested (ELISA, flow cytometry, and/orimmunocytochemistry) against a wider variety of targets, which includedcell lines, KG1a, K562, HT-29, and HEL 92.1.7, described above, andperipheral blood leukocytes (granulocytes, lymphocytes, monocytes, andthrombocytes). All clones with unique antibody profiles were thensubjected to limiting dilution cloning to produce a monoclonal hybridomasecreting an antibody of a single isotype and with a defined antigenspecificity.

Detection of Antibody-Positive Clones by a Sandwich-Enzyme LinkedImmunoadsorbent Assay (ELISA)

The sandwich ELISA technique used was modified from the method asdescribed by Harlow & Lane, “Chapter 14: Immunoassays,” in Antibodies: ALaboratory Manual, Harlow & Lane, eds., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988), pp. 553-612. Briefly, the ELISAwas run in U-shaped, 96-well microplates whose wells had been previouslyblocked, overnight, with 3% bovine serum albumin (BSA) in PBS, (pH7.5).Cells to be used as the antigen (NSC 1.1, KG 1a, K562, HT29, andHEL92.1.7) were washed three times in PBS, resuspended in PBS,enumerated (by trypan blue dye exclusion) and the cell number adjustedto 2×10⁶/ml. One hundred microliters (2×10⁵ cells/well) of these cellsuspensions were then added to each well of 96-well plates. All theplates containing the cells were subjected to centrifugation (2000 rpm,for 5 minutes at 4° C.) and the suprnatants from each of the wellsaspirated. Cells were resuspended in 100 μl of undiluted test hybridomasupernatant and incubated at 28° C. (room temperature) for 60 minutes.Each test hybridoma was run in triplicate wells. Following incubationwith the hybridoma supernatant, the cells were centrifuged (2000 rpm, 4°C., for 4 minutes) washed once with PBS and the cell pellets resuspendedin 50 μl of biotinylated rabbit/goat anti-mouse immunoglobulin (Ig)(VECTASTAIN Elite ABC Kit, Vecta Labs, Burligame, Calif.). The plateswere then re-incubated at 28° C. for a further 60 minutes. The cellswere again washed once in PBS and then resuspended in 100 μl ofVECTASTAIN ABC Avidin-HRP conjugate with a final incubation of 30minutes at 28° C. After incubation, the cells were washed once with PBSand the pellet resuspended in 100 μl of substrate-2,2 Azino-bis-3ethylbenz-thiazoline-6 sulphonic acid (ABT) (Sigma Chemicals Co, St.Louis, Mo.). The reaction was allowed to proceed for ten minutes, afterwhich the reaction was stopped. The assay was readspectrophotometrically at 405 nm and the data recorded. Results weredetermined to be positive if the O.D. reading was 2 standard deviationsabove the negative control.

Detection of Antibody-Positive Clones by Immunocytochemistry

The immunocytochemical procedure was as described in the manufacturer'sinstructions for the use of the VECTASTAIN ABC Elite Kit (Vecta Labs,Burligame, Calif.). Briefly, cells to be used (NSC1.1, KG1a, K562, HT29,HEL 92.1.7) were centrifuged (2000 rpm, 4° C., for 4 minutes) and thecells resuspended in PBS. The cells were washed a further 5 times in PBSand finally resuspended, in PBS, enumerated (Trypan blue dye exclusion)and the cell concentration adjusted to 1×10⁶/ml. Two hundred microlitersof each cell suspension were added to the cytospin chambers of acytocentrifuge and were then centrifuged onto glass slides (500 rpm for5 minutes). Once the cells were deposited onto the glass microscopeslides, they were air-dried then fixed in methanol at room temperature.Prior to testing hybridoma supernatants, the endogenous peroxideactivity was quenched by incubating the cell smears with a 1% solutionof hydrogen peroxide (H₂O₂) solution for one hour at room temperature.The slides were washed of excess H₂O₂ and then incubated for 60 minutesat room temperature with the undiluted test hybridoma supernatants.Following this step, the excess supernatant was removed, the slidesrinsed in 3×250 ml beakers containing fresh PBS, and each of the cellsmears incubated at room temperature for 30 minutes with 50 μlbiotinylated rabbit/goat anti-mouse Ig. Following the second antibodystep, the slides were again washed in PBS, as stated above, andincubated at room temperature for 30 minutes with 50 μl of Avidin-HRPconjugate, washed, then incubated for ten minutes with 100 μl of thesubstrate 3,3 diaminobenzidine (DAB). The reaction was stopped, airdried and the cells permanently fixed and mounted under Dpex and a glasscoverslip. All slides were then examined by light microscopy (usingeither 40× objective or 40× oil objective). The results were scored asfollows:

++++ Very strong positive reaction +++ Strong positive reaction ++Positive reaction + Weak positive reaction − Negative reactionDetection of Antibody Positive Clones by Flow Cytometry

For the detection of binding of specific antibody to the surface ofNSC1.1 cells, flow cytometry was carried out as described below.Briefly, staining of the NSC1.1 cells was performed on two-to-three dayold cell cultures. Cells were adjusted to a cell concentration of 1×10⁶cells per sample (200 μl) in IMDM containing 1% fetal bovine serum(FBS), L glutamine (0.292 mg/ml) and antibiotics (50 Units/mlpenicillin, and 50 Units/ml streptomycin). To the cells was added 100 μlof the test hybridoma supernatant, the mixture vortexed, and incubatedfor 30 minutes on ice. Following incubation, the samples were washed inthe above media and the cells resuspended in 200 μl of IMDM medium(shown above) and 50 μl of fluorescein isothiocyanate (FITC)-labeledgoat anti-mouse IgG (Fab)′2 (GAM-FITC). The labeled cells were thenincubated for 30 minutes on ice. After labeling, the cells were againwashed, resuspended in 1 ml of the above IMDM medium and then subjectedto flow analysis using an Epics Elite ESP (Coulter Corporation, Hialeah,Fla.) equipped with a 488 nm argon air-cooled laser. Forward and sidescatter gates were adjusted to include live cells only. Results of theanalysis were based on the collection of 10,000 data points per sample.

Separation of Peripheral Blood Leukocyte Sub-Sets by Self GeneratedPERCOLL Gradients

Subsets of peripheral blood leukocytes were separated using PERCOLL(Pharmacia, Piscataway, N.J.) according to the method described in themanufacturer's instructions. The self generating 70% PERCOLL (in 0.15MNaCI) gradients were generated by centrifuging 10 ml of PERCOLL solutionin 15 ml pyrex glass tubes (Coming, Cambridge Mass.), at 20,000×g (J20rotor) for 15 minutes at 10° C. Two ml of fresh citrated (ananticoagulant) peripheral blood was then overlaid onto the PERCOLLgradients and centrifuged at 800×g for 25 minutes at 10° C. Followingcentrifugation, the blood cells were separated according to theirdensities, i. e., platelets remained at the blood/PERCOLL interface,mononuclear cells (lymphocytes and monocytes) banded in the center ofthe gradient, and the polymophonuclear cells (granulocytes) anderythrocytes banded to the bottom of the gradients. Using a 5 ml pipettethe layers of discrete cells were harvested from the gradient, placed inseparate 15 ml polypropylene tubes, centrifuged (2000 rpm for 4 minutesat room temperature) and the pellets washed 3 times in IMDM (withoutsupplements). The final cell pellet was then resuspended in IMDM(without supplements) at the required cell concentrations for eitherimmuno-staining or western blot analysis.

Characterization of the Monoclonal Antibody Isotype

The identification of the monoclonal antibody isotype(s), being secretedby hybridomas of interest, was carried out using a mouse Ig typing kit(Pharmingen, San Diego, Calif.). The procedure was as described in themanufacturer's instructions for use. Briefly, 100 μl of rat monoclonals,with specificity for each mouse Ig isotype was aliquoted in wells (8wells/isotype) of a flat-bottomed microplate and incubated at 4° C.overnight. Following the overnight incubation, the supernatants fromeach well were aspirated, 300 μL of a 3% BSA solution (blocking agent)was added, and the plates incubated for a further 30 minutes at roomtemperature. Following this incubation step, the microplates were washed5 times with PBS/TWEEN 20 (0.5%) and 100 μl of test hybridomasupernatant added after the last wash. The plates were incubated for 60minutes at room temperature. After incubation, the plates were thenwashed, as previously described, and 100 μl of alkaline phosphataselabeled polyclonal rat anti-mouse Ig added, and the plates re-incubatedfor 60 minutes at room temperature. Prior to the substrate being added,the plates were washed 5 times in PBS/TWEEN 20. The substrate (p-N.P.)and its concentration used were as suggested in the isotyping kit.Following the color development, the results were determined using aspectrophotometer at a wave length of 405 nm.

Preparation of Cell Lysates for SDS-PAGE

Cells were harvested, centrifuged (2000 rpm at room temperature) andwashed 4 times in PBS. After the final wash, the cell pellets wereresuspended (vortexed) in lysis buffer (0.1% TRITON-X100 in PBS, and 1mM phenylmethyl-sulfonylfluoride (PMSF) and incubated on ice for 60minutes. After incubation, the cell lysates were clarified bycentrifugation (14,000 rpm at 4° C. for 45 minutes) and the supernatantcollected and used as a stock cell lysate preparation.

Protein Molecular Weight Determination by Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDS-PAGE)

The SDS-PAGE procedure used was as described by Laemmli, Nature227:680-685 (1970). Samples (cell lysates) were prepared for onedimensional SDS-PAGE by boiling the lysate for 5 minutes in the presenceof SDS and 2.5% β-mercaptoethanol. Protein samples were routinelyresolved using a premade 10% polyacrylamide gel (BioRad Inc., Richmond,Calif.). Gels were run at a constant voltage of 75V for approximately4-5 hours. Pre-stained SDS-PAGE molecular weight standards (BioRad Inc.,Richmond, Calif.) were routinely included on each gel. Electrophoresedgels were stained either using Coomassie blue-R250 (Sigma Chemicals, St.Louis, Mo.) or by silver stain (BioRad Inc., Richmond, Calif.). Theapproximate molecular weight of proteins of interest was calculatedusing regression analysis.

Detection of Antibody Ligands by Western Blot Analysis

Western blot analysis using the ECL detection system (Amersham,Arlington Heights, Ill.) was carried out as described by Boman et al.,Nature 358:512-514 (1992); Dalemans et al., Nature 354:526-528 (1991);Egan et al., Nature 358:581-584 (1992); and Kleijmeer et al., Nature357:342-344 (1992). Cell associated proteins were separated usingSDS-PAGE, as previously described. These SDS-PAGE gels were thenelectroblotted onto pre-blocked nitrocellulose membranes (BioRad Inc.,Richmond, Calif.). The blocking of nitrocellulose membranes wasaccomplished by overnight treatment at 4° C. of the membranes with 5%non fat dry skimmed milk powder dissolved in PBS plus 0.01% TWEEN 20.The electroblot and transfer of the proteins to the nitrocellulose wasperformed using a Tris-Glycine transfer buffer containing 0.1% SDS andwas carried out for 90 minutes at a constant voltage (100V). Followingtransfer, the nitrocellulose membranes were washed in a wash buffer(PBS, pH7.5, plus 0.01% TWEEN 20) and then incubated at room temperaturefor 60 minutes with primary antibody (1/10 dilution of hybridomasupernatant in 25 ml of blocking solution or 14 μg of purified antibodyin blocking solution). Following the incubation with the primaryantibody the membranes were washed in PBS/0.01% TWEEN 20 and thehorseradish peroxidase labeled goat anti-mouse IgG secondary antibodywas added (1/2000 dilution). The membranes were re-incubated at roomtemperature for 30 minutes then washed three times in PBS. Detection ofantigen/antibody complexes was performed using the chemiluminescentreagent from Amersham's ECL system. The resulting X-ray films weredeveloped using a Kodak M35A X-OMAT processor and the autroradiographsexamined for positive reactions.

Determination of Protein Glycosylation

The presence/absence of carbohydrate moieties on the MG1 antigen wasassessed by enzyme deglycosylation of the protein backbone. In thisassay, digestion of any sugar present results in a shift in molecularweight that can be visualized by SDS-PAGE and western blot analysis.Deglycosylation of the MG1 antigen was carried out using the followingenzymes: for O-linked sugar determination, O-glycosidase (BoehringerMannheim, Indianapolis, Ind.), for N-linked sugar determination,N-glycosidase (Boehringer Mannheim, Indianapolis, Ind.). Briefly, NSC1.1lysate (50 μg total protein) was aliquoted into 1.5 ml microfuge tubesand to these tubes was added either N-glycosidase (6 Units/50 μgprotein), O-glycosidase (25 mUnits/50 μg protein) or a combination ofthe two. A microfuge tube containing 50 μg of protein only (no enzymes)was used as an untreated control. All of the tubes were incubated for 24hours in a 37° C. waterbath. Following incubation, 100 μl of 2× SDSreducing sample buffer was added to all tubes, and then placed in aboiling waterbath for 5 minutes. The samples were run on a SDS-PAGE (aspreviously described), electroblotted onto a nitrocellulose filter, andthen probed using the MG1 monoclonal antibody (as described above).

Purification of Mouse Monoclonal Immunoglobulins by AffinityChromatography

The purification of immunoglobulins from 3.2B11.b3 hybridomasupernatants, using affinity-based chromatography, was as described bySchwartz, L., “Use of immobilized protein A to purify immunoglobulins,”in Bacterial Immunoglobulin-Binding Proteins: Applications inImmunotechnology, M. D. P. Boyle, Academic Press (1990), pp.309-339, andWalker, W. B., “Use of immobilized protein G to isolate IgG,” inBacterial Immunoglobulin-Binding Proteins: Applications inImmunotechnology, M. D. P. Boyle, Academic Press (1990), pp. 355-368.Briefly, stock cultures of the hybridoma were centrifuged (2000 rpm, for4 minutes at room temperature), then resuspended in fresh IMDMsupplemented with 5% FBS (Ultra-low IgG, Life Technologies), and placedin T₁₅₀ cm² tissue culture flasks and grown at 37° C., in a humidifiedatmosphere of 5% CO₂ in air. Once the hybridoma reached confluency,supernatants from the cultures were harvested, centrifuged (2000 rpm for10 minutes at room temperature), and then sterilized using a 0.22 μmfilter (Amicon, Beverly, Mass.). Five ml HITRAP protein G columns(Pharmacia Biotech, Piscataway, N.J.) were equilibrated with 20 ml of 20mM sodium phosphate buffer (pH 7.0). Approximately 500 ml of hybridomasupernatant was loaded using a peristaltic pump, onto eachpre-equilibrated column at the flow rate of 1 ml/min. The column wasthen placed in line on a Pharmacia Fast Protein Liquid Chromatography(FPLC) system and again washed with 20 mM sodium phosphate, buffer (pH7.0) until the UV detector/chart recorder returned to baseline. Thebound IgG was then eluted with 10-15 ml of 0.1M glycine buffer (pH 2.7)at a flow rate of 1 ml/minute and collected as a single fraction in atube containing 200 μl of 1M Tris-HCL (pH 9.0).

The purified IgG fraction was desalted against 20 mM sodium-phosphatebuffer (pH 7.0) using PD-10 gel filtration columns (Pharmacia Biotech,Piscataway, N.J.) as per manufacturer's instructions. Concentration ofthe purified IgG was carried out using a Centriplus 10 (Amicon) andcentrifuged at 3000×g for 60 minutes. Protein concentration wasdetermined by Bradford protein assay and SDS-PAGE gels were run toascertain the purity of the immunoglobulin preparation. The antibody wasthen stored at 4° C. or −20° C.

Purification of MG1 Antigen by Affinity Chromatography

Purification of the cell surface antigen or ligand recognized by the MG1monoclonal antibody was carried out by immuno-affinity chromatography aspreviously described (Harlow & Lane, “Chapter 13: ImmunoaffinityPurification,” in Antibodies: A Laboratory Manual, Harlow & Lane, eds.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988),pp.511-552). Purified MG1 IgG was covalently coupled to the sepharosematrix of a HITRAP NHS-activated 5 ml column (Pharmacia Biotech,Piscataway, N.J.) as per manufacturer's instructions. Briefly, thecolumn was connected to a peristaltic pump and washed with 30 ml ofice-cold 1 mM HCL at a flow rate of 2 ml/min. Twelve mg purified MG1 IgG(in 25 ml of 0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3) was then pumped onto thecolumn at the flow rate of 1 mmin. The MG1 containing solution wasrecirculated and pumped back over the column. The column was incubatedfor 30 minutes at room temperature (25° C.). Excess active groups weredeactivated by washing the column with 0.5 M ethanolamine, 0.5 M NaCl,pH 8.3 and non-specifically bound ligands were eluted off the columnwith 0.1 M acetate, 0.5 M NaCl, buffer (pH 4.0).

The MG1-immuno-affinity column was equilibrated with 30 ml of 20 mMsodium-phosphate (pH 7.0). Ten ml of NSC1.1 cell lysate (prepared asstated above) was loaded into a 10 ml FPLC superloop (Pharmacia Biotech,Piscataway, N.J.) and injected onto the column at the flow rate of 0.5mmin. The column was then washed with 20 mM sodium-phosphate, (pH 7.0)until no UV absorbance was recorded in the flow-through. The boundligands were eluted with 10 ml of 0.1M glycine (pH 2.7) and collected asa single fraction in a tube containing 200 μl of 1 M Tris-HCl, (pH 9.0).

The eluted fraction containing the MG1 antigen was desalted against 20mM sodium-phosphate buffer, (pH 7.0) using PD-10 gel filtration columns(Pharmacia Biotech, Piscataway, N.J.) as per manufacturer'sinstructions. Concentration of the purified MG1 antigen was carried outusing a Centriplus 10 (Amicon) and centrifuged at 3000× g for 60minutes. Purity of the MG1 antigen and its apparent molecular mass wasassessed by SDS-PAGE and Western blotting.

Determination of the Amino Acid Composition of the MG1-Antigen

The amino acid composition of the MG1-antigen was determined as follows.The MG1-antigen was purified by immunoaffinity column purification,followed by SDS-PAGE and transfer of the ligand band to PVDF membranes.The amino acids were quantitatively released, without degradation, byhydrolysing 0.45 μg of the purified MG1-antigen with 6N HCl for 24 hoursat 110° C. under vacuum. After hydrolysis, the sample was dried andreconstituted for analysis.

Amino acid analyses were performed on the Applied Biosystems Model 420AAnalyzer, an automated PTC amino acid analysis system. Briefly, thesample was applied to a glass frit support, which delivered the sampleto a flow-through reaction chamber where the amino acids werederivatized with PTC using 5% phenylisothiocyanate in heptane. Afterderivatization, the PTC-amino acids were transferred on-line to an HPLCwhere each of the PTC-amino acids is separated based on its retention ona reverse phase C18 HPLC column. Amino acid composition is determinedwith an overall error of approximately 10%, however, by restrictingfurther analyses to specific amino acids that give more reliableresults, it is possible to improve the accuracy. The amino acidcomposition data was used to attempt to identify the protein bycomparison to the available protein databases using the ExPASy,(Wilkins, M. R. et al., Bio/Technology 14:61-65 (1996), and Wilkins, M.R. et al., Biochem. Biophys. Res. Commun. 221:609-613 (1996)), andPropsearch (Hobohm, U., et al., Analytical Biochemistry 222:202 (1994))programs.

Detection of Laminin Reactivity by Western Blot Analysis

The reactivities of the MG1 antibody against mouse laminin, and of theMG1-antigen with anti-laminin polyclonal antibodies, were determined bywestern blot, essentially as described above. Briefly, NSC1.1 lysate,NSC1.1 conditioned medium, and purified mouse laminin, were run intriplicate on SDS-PAGE and transferred to nitrocellulose membranes asdescribed. Following transfer, the nitrocellulose membranes wereseparated into three parts and the individual parts were washed in awash buffer (PBS, pH7.5, plus 0.01% TWEEN 20) and then incubated at roomtemperature for 60 minutes with primary antibody. The membranes werelabeled with anti-laminin polyclonal antibody (rabbit serum, againsthuman placental laminin, Chemicon International Inc.; lot #58296268) ata 1:2000 dilution, or MG1 monoclonal antibody (1/10 dilution ofhybridoma supernatant in 25 ml of blocking solution or 14 μg of purifiedantibody in blocking solution), or PBS alone. Following incubation withthe primary antibody, the membranes were washed in PBS/0.01% TWEEN 20and horseradish peroxidase labeled goat anti-mouse (or donkeyanti-rabbit) IgG secondary antibody was added (1/2000 dilution). Themembranes were re-incubated at room temperature for 30 minutes, thenwashed three times in PBS. Detection of antigen/antibody complexes wasperformed using the chemiluminescent reagent from Amersham's ECL system.The resulting X-ray films were developed using a Kodak M35A X-OMATprocessor and the autroradiographs examined for positive reactions.

EXAMPLE 1

Characterization of the MG1 Monoclonal Antibody

The Derivation of the 3.2.B11.b.3 Hybridoma Subclone

The three mice immunized with the NSC1.1 subclone (NSC-1.1) weresacrificed and their spleens used in fusion experiments for hybridomaproduction. Use of the spleens for fusion was based upon the reactivity,as measured by both flow cytometry and immunocytochemistry, of theindividual mouse sera with NSC1.1 cells. The sera from all three micegave very strong reactions using both detection systems.

The origin of the NSC1.1-specific hybridoma subclone (3.2.B11.b3) isshown in FIG. 1. Due to bacterial contamination of the mouse#2spleen/SP2/0 fusion culture, no clones developed. From the remaining twomice (#1 and #3), 178 hybridomas (polyclones) were recorded.Supernatants removed from all 178 hybridomas were tested utilizing anindirect ELISA. Thirty of these hybridomas gave strong positivereactions against the NSC1.1 cell line. Over a period of two weeks, the30 hybridoma cultures were expanded and continually monitored forreactivity to the NSC1.1 cell line. During this period, eight hybridomaswere lost. The ELISA screening of the remaining 22 clones was alsoexpanded to include erythrocytes as an antigen. Clones that hadsignificant reactivity to both NSC1.1 cells and erythrocytes were thendiscarded. From these experiments, only 10 hybridoma clones gavepositive reactions against NSC1.1 cells alone. Following further passageof these 10 clones, 4 clones were lost. The remaining 6 clones wereexpanded to T75 cm² tissue culture flasks and samples from each storedin liquid nitrogen. Verification of NSC1.1 reactivity by all 6 cloneswas assessed by immunocytochemistry. Three clones with the strongestreactivity (3.2.B2, 3.2.B11, and 1.3.D9) were then subcloned by limitingdilution to obtain single cell hybridomas. All attempts to subclone 1.3D9 to obtain single cell clones failed. Three monoclones were generatedfrom the limited dilution cloning of 3.2.B2 and 7 monoclones weregenerated from the limited dilution cloning of 3.2.B11, one of which was3.2.B11.b3. This clone was subsequently shown to secrete a monoclonalantibody of unique specificity and was designated “MG1.”

Purification and Isotype Identification of the Monoclonal AntibodySecreted by the Hybridoma3.2.B11.b3 (MG1)

The monoclonal antibody secreted by the 3.2.B11.b3 hybridoma waspurified to homogeneity using protein-G affinity chromatography. Theprotein eluted from the column was subjected to SDS-PAGE electrophoresisto ascertain the purity of the preparation. The photomicrograph of aCoomasie blue-stained SDS-PAGE of the eluted protein, at differentconcentrations (FIG. 2), shows two distinct bands; one protein band atapproximately 60 kDa (an equivalent molecular weight to the heavy chainof an Ig) and the other at approximately 28 kDa (an equivalent molecularweight to the light chain of Ig). There is no evidence of otherprotein(s) in the purified preparation.

Identification of the mouse immunoglobulin isotype for the MG1 antibodywas carried out using the Pharmingen isotype typing kit, the results ofwhich are shown in Table 1.

TABLE 1 ISOTYPE OF MG1 IMMUNOGLOBULIN Anti-isotype positive negative AbMG1 IgG MG1 IgG control control Anti-IgG 1 0.019 0.025 0.768 0.023Anti-IgG 2a 0.871 0.808 0.435 0.000 Anti-IgG 2b 0.034 0.038 2.725 0.000Anti-IgG 3 0.029 0.035 0.208 0.000 Anti-IgM 0.027 0.076 1.073 0.000Anti-IgA 0.026 0.027 0.597 0.010 Anti-Ig Lκ 2.452 2.373 1.147 0.004Anti-Ig Lλ 0.020 0.019 0.807 0.000

The isotype of the purified MG1 immunoglobulin was determined by ELISA.Plates pre-coated with anti-isotype antibody were blocked with 3% BSA inPBS, before the addition of the MG1 or control immunoglobulins. Alkalinephosphatase tagged rat anti-mouse Ig was used as the secondary antibody.Reactions were read spectrophotometrically at 405 nm. Background levelsof absorbance were determined for each row using wells treated with onlythe immobilized anti-isotype antibody, the enzyme tagged secondaryantibody, and enzyme substrate. The positive control used was mouseimmunoglobulin cocktail. The negative control used was mouse myeloma(Sp2/014) supernatant. The data shows that the MG1 isotype is murineIgG2a.

The Reactivity of Clone 3.2.B11.b3 (MG1) with Established Cell LinesDerived from Hematopoietic Malignancies. HT-29 Cells. and Normal HumanBone Marrow

Given the fact that the NSC1.1 cell line was derived from a humanhematopoietic stem cell population (CD34⁺), it was likely that the MG1antibody would recognize stem cells and/or immature (progenitor) cellsof the hematopoietic system. To test this, flow cytometry was performedon tumor cell lines isolated from malignancies of early stem cells,namely KG1a (an acute myelogenous leukemia), K562 (a chronic myelogenousleukemia), and HEL 92.1.7 (an erythroleukemia), and on normal human bonemarrow. From the flow cytometric analysis data (FIG. 3), it is evidentthat the MG1 monoclonal antibody, while recognizing 96.9% of NSC1.1cells, does not recognize any of the cells representing these threeleukemias. It is interesting to note that KG1a was the cell line used todiscover the CD34 antigen and since MG1 does not recognize KG1a, it ishighlyprobable that MG1 does not recognize the CD34 antigen. Further,the MG1 monoclonal antibody did not label HT-29 cells. Thisnon-hematopoietic cell line is derived from a human colonadenocarcinoma. These results suggest that MG1 is not recognizingantigens present on non-hematopoietic tissues.

Using fluorescein-labeled MG1 monoclonal antibody, whole human bonemarrow was analyzed by flow cytometry. The data from FIG. 4 givesfurther evidence that MG1 recognizes only a very small population (lessthan 1%) of cells within bone marrow. Furthermore, live-gating on CD34⁺cells indicated that less than 3% of CD34⁺ cells co-express MG1 antigen,suggesting a very small overlap of the two populations.

Utilizing immunomagnetic bead cell separation technology and purifiedMG1 antibody, MG1⁺ bone marrow cells were recovered. When thecolony-forming potential of the MG1⁺-selected and the MG1-depletedpopulations were compared in CFU-GEMM assays, it was observed that theMG1⁺ cells were nearly 300-fold poorer at forming colonies than theMG1-depleted population (0.23 colonies per 10⁵ cells vs. 70±17 coloniesper 10⁵ cells, respectively. The lack of CFU activity exhibited by theMG1 cells with recombinant human IL-3, Il-6, and Epo in CFU-GEMM assaysgave further evidence to the primitive nature of the MG1⁺ cellpopulation. It is well established that quiescent hematopoietic stemcells, while retaining high proliferating potential, fail to respond toexogenous cytokines in CFU assays. (Berardi et al., Science 267:104-108(1995)). These cells however, will respond and proliferate in ex vivolong term culture initiating cell (LTCIC) assays. Berardi et al.,Science 267:104-108 (1995); Id.; Young et al., Blood 87:545-556 (1996).

Analysis of Specificity of the MG1 Monoclonal Antibody by Western Blot

Western blot analysis was performed with two purposes in mind: First, tounderstand the scope of MG1's reactivity on different cell populationsand second, to identify the ligand to which MG1 binds. Using lysatesprepared from the leukemia cell lines (KG1a, K562, and HEL 92.1.7),western blot analysis was carried out, the results of which are shown inFIG. 5 (western blot using MG1 hybridoma supernatant—1/10 dilution),FIG. 6 (western blot using MG1 hybridoma supernatant—1/100 dilution) andFIG. 7 (western blot using purified MG1 IgG2a). The data from FIGS. 5-7demonstrate that the only reactivity obtained was MG1 binding to theNSC1.1 lysate (lanes 2 and 9 of FIG. 5, lanes 1 and 9 of FIG. 6, andlanes 2 and 9 of FIG. 7). These results confirm the earlier flow data(FIG. 3) in which the MG1 monoclonal antibody only bound to the surfaceof the NSC1.1 cell and not to any of the cell lines mentioned above. Thesecond observation to be made from FIGS. 5-7 is that the protein that isrecognized by MG1 is a high molecular weight protein with a calculated(regression analysis) molecular weight of approximately 186 kDa±5%.Finally, a comparison of FIGS. 6 and 7 show that the hybridomasupernatant and the purified IgG from the monoclonal hybridoma recognizethe same protein in Western analyses. This confirms the derivation ofthe monoclonal.

Similar western blot experiments were carried out using cell lysatesfrom subpopulations of peripheral blood leukocytes enriched on selfgenerated PERCOLL gradients. The blots were again probed with purifiedMG1 monoclonal antibody (14 μg). The data from FIG. 8 conclusively showthat the MG1 antibody probe only recognizes NSC1.1 lysate (lanes 1 and10) and does not recognize proteins associated with cells isolated fromnormal peripheral blood. Again, the protein recognized by MG1 antibodyappears to have a molecular weight of approximately 186 kDa±5%.

Therefore, combining all the data from the western blot analyses and theflow cytometry together, it is believed that the MG1 monoclonal antibodyrecognizes a cell surface antigen that is only expressed on rare cellswithin the hematopoietic environment. Furthermore, given the CFU data,it is believed that these cells reside within the primitive stem cellcompartment of that tissue.

EXAMPLE 2

Characterization of the MG1 Antigen

The MG1 antigen is expressed on a small proportion of human bone marrowcells, and this population overlaps with the CD34⁺ population. MG1 andCD34 doubly labeled cells represented only minor subpopulations of thesingly labeled cells. The results of flow analyses on bone marrowindicate that this overlapping population represents a small percentageof the CD34⁺ population, and also a small percentage of the MG1⁺population. In other words, few of the CD34 population also labels withMG1, and few of the MG1 population also labels with CD34. The fact thatindependent flow studies showed that MG1 did not recognize any of thesubpopulations of the peripheral blood leukocytes enriched on selfgenerated PERCOLL gradients, suggests that MG1 is not recognizing maturecells. Furthermore, when MG1-selected cells are placed into CFU-GEMMassays (with cytokines IL-3, IL-6, SCF and Epo), few if any colonies aredetected, whereas the MG1-depleted population does generate colonies.This indicates that MG1 is not recognizing committed progenitor cellseither. Recent results showing that high proliferative potential ofhematopoietic cells is associated with quiescence in CFU assays (Berardiet al., Science 267:104-108 (1995); Young et al., Blood 87:545-556(1996)) would suggest that the MG1⁺ population falls into the categoryof very primitive, quiescent cells.

Glycosylation Determination of the MG1 antigen In order to characterizethe MG1 antigen more fully, the protein was subjected to deglycosylationusing glyconases specific for N-linked and O-linked carbohydratemoieties. It is evident from the data shown in FIG. 9 that the MG1antigen is glycosylated and further, that this glycosylation ispredominantly comprised of N-linked sugars (lane 2). Treatment of theMG1 antigen with O-glyconase did not measurably (as detectable by theresolving capability of this gel) decrease the molecular weight of theligand (lane 3) indicating the lack of O-linked sugars. This was furtherconfirmed by treatment of the MG1 antigen with a mixture of the twoenzymes, the results of which are shown in lane 1 of FIG. 9, wherein thedecrease in the molecular weight observed is equal to that of theN-glyconase treatment alone.

Purification of the MG1 antigen The MG1 antigen was partially purifiedby affinity column chromatography. Lysates prepared from NSC1.1 cellswere loaded onto an immunoaffinity column containing bound purified MG1monoclonal antibody. The column was washed, and the bound protein waseluted with 0.1 M glycine (pH 2.7) into 1.0 M Tris (pH 9.0). This eluatewas concentrated by ultrafiltration, and examined by SDS PAGE. The sizeof the MG1 antigen, as calculated by a regression analysis of the twoprotein marker lanes in FIG. 10 (non-prestained), was about 186 kDa±5%.

Reactivity with Laminin The amino acid composition of the purifiedligand was determined (see Table 2) and compared to other previouslydescribed proteins in available databases using two different computerprograms (ExPASy and Propsearch).

TABLE 2 AMINO ACID COMPOSITION FOR MG1 ANTIGEN Amino Acid Percent ofTotal Asx 12.81 Glx 15.47 Ser 5.34 His 3.37 Gly 11.64 Thr 4.34 Ala 6.92Pro 5.43 Tyr 2.56 Arg 4.12 Val 6.06 Met 0.71 Ile 4.92 Leu 6.85 Phe 3.17Lys 6.29Results indicated that the MG1 antigen appeared to be a unique and novelprotein. However, both programs found a low degree of similarity with β1subunit of human laminin. In order to examine the possibility that theMG1 antigen could be the β1 subunit of human laminin, a Western blotanalysis was conducted using NSC1.1 lysate and a rabbit polyclonalantibody against human laminin (FIG. 11). The results indicated that theanti-laminin antibody did not recognize any proteins in the NSC1.1lysate suggesting that the MG1 antigen is not the β1 subunit of humanlaminin.

N-terminal amino acid sequencing of purified MG1 antigen was alsoperformed. The partial amino acid sequence of the MG1 antigen is shownbelow (in standard three letter amino acid code):ArgArgArgAlaLysGlnAsnGlnXaaGlyGluIle  (SEQ ID NO. 1)(where Xaa represents an undetermined residue). Since no otherpreviously described proteins showed significant sequence identity withthe amino acid composition and sequence of MG1 antigen, it was concludedthat the MG1 antigen represents a novel protein.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. An isolated polynucleotide molecule that encodes a polypeptide, or a fragment thereof, expressed on the surface of a subset of human CD34+ hematopoietic mononuclear cells, wherein said polypeptide irnmunoreacts with MG1 antibody, wherein said MG1 antibody is produced by the hybridoma cell line deposited under ATCC Accession No. HB12232, and wherein said polypeptide comprises the amino terminal amino acid sequence of SEQ ID NO:
 1. 2. The polynucleotide according to claim 1, wherein said polypeptide exhibits a molecular weight of about 186 kDa as determined by SDS-PAGE.
 3. The polynucleotide according to claim 1, wherein said polypeptide has an amino acid composition as shown in Table
 2. 4. An isolated polynucleotide molecule that encodes a polypeptide, or a fragment thereof, expressed on the surface of a subset of human CD34+ hematopoietic mononuclear cells, wherein said polypeptide immunoreacts with MG1 antibody, wherein said MG1 antibody is produced by the hybridoma cell line deposited under ATCC Accession No. HB12232, wherein said polypeptide comprises the amino terminal amino acid sequence of SEQ ID NO: 1, wherein said polypeptide exhibits a molecular weight of about 186 kDa as determined by SDS-PAGE, and wherein said polypeptide has an amino acid composition as shown in Table
 2. 